Method and system for imaging high density biochemical arrays with sub-pixel alignment

ABSTRACT

A method and associated system for imaging high density biochemical arrays comprises one or more imaging channels that share a common objective lens and a corresponding one or more time delay integration-type imaging cameras with optical alignment mechanisms that permit independent inter-channel and intra-channel adjustment of each of four degrees: X, Y, rotation and scale. The imaging channels are configured to independently examine different spectra of the image of the biochemical arrays.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/856,369, filed on Apr. 3, 2013, titled “Method and System for ImagingHigh Density Biochemical Arrays with Sub-Pixel Alignment,” which is acontinuation of U.S. application Ser. No. 13/451,678, filed on Apr. 20,2012, titled “Method and System for Imaging High Density BiochemicalArrays with Sub-Pixel Alignment,” now U.S. Pat. No. 8,428,454, which isa continuation of U.S. application Ser. No. 12/912,641, filed on Oct.26, 2010, titled “Method and System for Imaging High Density BiochemicalArrays with Sub-Pixel Alignment,” now U.S. Pat. No. 8,175,452, thecontents of which are incorporated herein by reference in theirentirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

The disclosure is generally related to the field of imaging systems forhigh-density biochemical arrays.

High-density biochemical arrays and associated machines allow multiplebiochemical experiments, sometimes billions, to be performed inparallel. This ability accrues from the development of techniques toperform each experiment in a very small volume and to pack theexperiments very close together. To observe the experiments efficiently,advances analogous to miniaturization advances in other high technologyindustries are needed. Specifically, what is needed are fast, accurate,repeatable and robust imaging techniques for biochemical arrays.

SUMMARY

According to the invention, a system and associated method for imaginghigh density biochemical arrays comprises one or more imaging channelsthat share a common objective lens and a corresponding one or more timedelay integration-type imaging cameras with optical alignment mechanismsthat permit independent inter-channel and intra-channel adjustment ofeach of four degrees of freedom: X, Y, rotation and scale. The imagingchannels are configured to independently examine different wavelengthsin the image of the biochemical arrays.

The invention will be better understood by reference to the followingdetailed description in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of a first multichannel biochemical array imagingsystem.

FIG. 1B is a diagram of a second multichannel biochemical array imagingsystem.

FIG. 1C is a diagram of a third multichannel biochemical array imagingsystem.

FIG. 1D is a diagram of a fourth multichannel biochemical array imagingsystem.

FIG. 2 is a diagram of lateral offset plate.

FIG. 3 illustrates X and Y offsets.

FIG. 4 illustrates X and Y alignment errors.

FIG. 5 is a conceptual diagram of imaging a spot with a pixel arrayusing time delay integration.

FIG. 6 is a conceptual diagram of results of the time delay integrationimaging of FIG. 5.

FIG. 7 shows rotational alignment relationships between two cameras, aslide and a positioning stage before alignment.

FIG. 8 shows rotational alignment relationships between two cameras, aslide and a positioning stage after alignment.

FIG. 9 illustrates reference frames used in slide alignment.

DETAILED DESCRIPTION

Human genome studies and other uses of biochemical arrays requireadvanced imaging systems to achieve commercially viable data acquisitionrates. The number of biochemical experiments from which data may becollected per unit time depends on array density and image acquisitionspeed among other factors. Increased array density complicates the imageacquisition problem because it makes keeping track of the identity ofeach experiment (out of millions) in an image challenging.

For DNA arrays the desired data are usually quaternary; a nucleotide maybe A, C, G or T. These possibilities are labeled with a set of fourdifferent colored fluorescent molecular tags. Each fluorescent tagabsorbs light of a certain wavelength and emits light of a longerwavelength. A multichannel imager collects data in as many of the fourpossible wavelength bins as possible simultaneously.

FIGS. 1A-1D are diagrams of multichannel biochemical array imagingsystems. Each imaging channel in these multichannel systems has its ownindependent adjustments for image rotation, x and y offset, and scale(magnification), as hereinafter explained as intrachannel andinterchannel adjustment independence. FIG. 1A illustrates a two-channelsystem. FIGS. 1B and 1C illustrate the system of FIG. 1A with alternatemeans of adjusting x and y image offset. FIG. 1D illustrates howmultiple four-degrees-of-freedom imaging channels may be added to amultichannel system.

The system of FIG. 1A has two simultaneous imaging channels each withfour degrees of freedom for image adjustments: rotation, x and y offset,and scale or magnification. A high precision positioning stage scans aslide under a microscope objective lens that is characterized by an axisof rotational symmetry.

In FIG. 1A, conventional time delay integration (TDI) camera 105 ismounted on rotation stage 102. Camera 105 may operate in TDI mode orfull frame mode depending on what operations the system is performing.Lateral offset plate 110 shifts the position of an image in camera 105.Tube lens 117 and helper lens 115 together form a zoom lens system forfocusing and changing the size of an image in camera 105. The rotationstage 102 is configured to rotate the TDI camera 105 around a commonaxis of rotational symmetry 106 to orient the internal CCD array (notshown) of the TDI camera 105 with respect to a sample 145 so that thatthe sample 145 can be properly scanned along a scanning axis 104(through the plane of the figure). The camera 105, the rotation stage102, the plate 110 and the zoom lens system formed of tube lens 117 andhelper lens 115 together form one independent imaging channel 139. Asecond independent imaging channel 140 comprises a second camera mountedon a rotation stage, an offset plate and a zoom lens system. Beamsplitter and filter assembly 127 directs different wavelengths of lightto the different imaging channels 139, 140. Only one beamsplitter/filter assembly 127 is shown in FIG. 1A. However, in otherembodiments of the system, additional beam splitters and/or filters maybe moved in and out of the machine system by mechanical robots.Autofocus and illumination systems are represented by block 125.Microscope objective 130 common to all imaging channels is focused on asample 145 in the form of a biochemical array slide that is mounted on apositioning stage comprising rotation stage 135 and X-Y stage 137.

Light emitted by fluorescently tagged biomolecules is collected by themicroscope objective and focused onto pixels in one or the other of theTDI cameras, depending on wavelength. A system with two imaging channelscan record image data in two wavelength bins simultaneously.Substitution of different dichroic or polychroic beam splitters and/orfilters 127 allows image data to be collected in additional wavelength“bins.” Each imaging channel has its own zoom lens system to adjustimage focus and magnification. Such adjustments are typically made whenchanging dichroic filters, for example. Each camera may be independentlyrotated and the array slide may also be rotated on top of its X-Ypositioning system.

The zoom system is atypical in that it provides a constrained and verylimited range of magnification (scale) adjustment, but does so with veryhigh precision and stability. Lenses 115 and 117 are mounted onprecision stages (not shown) that move them along the lens axes in onemicron steps. In an example system the focal lengths f₁ and f₂ are about500 mm and 165 mm, respectively with the maximum change in scale notexceeding 3%. This precise zoom system allows the magnification of anominally 16× microscope to be adjusted in steps as small asapproximately 0.00009× while maintaining focus.

FIG. 1B shows a variation of the system of FIG. 1A. In FIG. 1B mirror111 replaces offset plate 110 of FIG. 1A. The mirror provides analternate means of offsetting an image in camera 105. Second imagingchannel 140 is not shown in FIG. 1B for clarity of illustration.

FIG. 1C shows a variation of the systems of FIGS. 1A and 1B. In FIG. 1Ccamera 105 is mounted on x-y positioning stage 103 as well as rotationstage 102. Neither an offset plate (e.g. 110 of FIG. 1A), nor an offsetmirror (e.g. 111 of FIG. 1B) are included in one of the channels of thesystem of FIG. 1C. Rather, mechanical x-y positioning stage 103 provideslateral offset control for camera 105.

FIG. 1D shows how systems like those illustrated in FIGS. 1A-1C may beconstructed with any number of imaging channels, each with parametersadjustable independently of one another and each channel beingadjustable independently of any other channel so that adjustments in onechannel have no effect on other channels. This intrachannel andinterchannel adjustment independence is herein denoted asfour-degrees-of-freedom imaging channel independence. In FIG. 1D beamsplitter/filter assemblies 128 and 129 direct different wavelengths oflight to imaging channels 141 and 142 respectively. Each imaging channelmay contain independent adjustments for image rotation, x and y offset,and scale or magnification. X and y offset control may be achieved withtilting plates (e.g. plate 110), mirrors (e.g. mirror 111), time delayintegration pulse timing (as described below) or a combination oftechniques. The lateral offset plate is described in more detail inconnection with FIG. 2.

FIG. 2 is a diagram of lateral offset plate 110. Plate 110 shifts theposition of images in camera 105. In FIG. 2 light beam 150 is shownpassing through plate 110 and emerging as light beam 152. Because thebeam passes through the plate at non-normal incidence, its position isoffset by an amount Δx given by:

${\Delta\; x} = {{t\;\sin\;\theta} - {\frac{t}{n}\frac{\cos\;\theta\;\sin\;\theta}{\sqrt{1 - \left( {\frac{1}{n}\sin\;\theta} \right)^{2}}}}}$where t is the thickness of the plate, n is its index of refraction andθ is the angle of incidence. A typical glass (n˜1.5) plate that isapproximately 2.5 cm in diameter and 3.5 mm thick weighs only a fewgrams and may be mounted on a galvo rotation mechanism for quick andprecise movements. A five degree tilt produces an offset of about 100μm.

Images may be shifted in the perpendicular (i.e. Y) direction relativeto the X axis through the use of time delay integration (TDI) pulsetiming in camera 105. FIG. 3 illustrates X and Y offsets. Spot 205 is aspot of light imaged on an array of pixels 210. Arrows indicate how thespot may be moved with respect to the pixel array. As described above, Xoffsets are adjusted by a galvo and offset plate system, while Y offsetsare adjusted by TDI pulse timing. In time delay integration, an image isscanned across pixels in a camera at (nominally) the same rate thatimage data is read out of the pixels. Slight changes in the dataread-out rate (or scan rate, or both) in effect shift the position ofrecorded images along a first axis, while slight changes in the angle ofthe galvo-controlled offset plate around the first axis can shift theposition of recorded images along the axis normal to the first axis.Thus the combination of TDI cameras having adjustable timing andgalvo-controlled offset plates offers a quick and precise way tointroduce independent, two-dimensional, lateral offsets in imagesrecorded by cameras in the imaging channels of a multichannel imagingsystem. Furthermore this method of introducing image offsets does notdepend on moving a slide with respect to an objective lens.

The galvo-controlled plate and TDI offset system just described areuseful for making small corrections to align an image of a biochemicalarray with an array of pixels in a camera. (The control mechanism isbeyond the scope of this disclosure.) FIG. 4 illustrates X and Yalignment errors between a spot 305 in an image and an array of pixels315. In FIG. 3, dotted circle and plus sign symbol 310 denotes thecenter of a pixel. The symbol comprising a solid circle and plus sign305 indicates the actual position of a spot in an image. “Δx” and “Δy”show the difference between positions 305 and 310. In one particularsystem, each 8 μm by 8 μm camera-based pixel images and thus correspondsto a 500 nm by 500 nm area of a biochemical array. It has been foundthat an imaging system such as the one illustrated in FIG. 1 canmaintain alignment to a biochemical array with better than 20 nmaccuracy while scanning more than one million data spots per second.

Achieving high throughput with high density arrays depends in part onaccurate mechanical scanning stages. In principle X-Y stage 137 in FIG.1 can move in any direction in the X-Y plane. Diagonal movement iscreated by a combination of X and Y movements. In practice, however,stage accuracy is best if one dimension (e.g. X) is fixed whilemovements in the other dimensions (e.g. Y) are taking place.

Similarly, time delay integration cameras achieve highest precision whenthey are scanned parallel to the direction of data read-out. FIG. 5 is aconceptual diagram of imaging a spot with a pixel array using time delayintegration. Misalignment causes image smearing as shown in FIG. 6 whichis a conceptual diagram of results of the time delay integration imagingof FIG. 5.

Spot 405 is imaged by array of pixels 415. The relative motion of thespot and the pixel array is shown by the dotted arrow originating atspot 405. The arrow is not aligned with the pixel array and smearedimage 420 is the unfortunate result. Spot 410 is also imaged by array ofpixels 415, but this time the relative motion of the spot and the pixelarray is shown by the dotted arrow originating at spot 410. The arrow isaligned with the pixel array and image 425 results.

Practical limitations of positioning stages and camera time delayintegration systems highlight the utility of providing each camera, andthe slide X-Y stage, with rotation stages. FIGS. 7 and 8 show rotationalalignment relationships between two cameras, a slide and a positioningstage. If one of these four elements is considered to be fixed, threedegrees of rotational freedom are required to align the other threeelements.

In FIGS. 7 and 8, X and Y axes 505 represent the orientation of a stagesuch as X-Y stage 137 in FIG. 1. The orientation of cameras in the first(e.g. camera 105 in FIG. 1) and second imaging channels are representedby 510 and 520 respectively. The orientation of a slide, such as slide145 in FIG. 1, is represented by 525. In FIG. 7 the two cameras, theslide and the stage are all rotationally misaligned with respect to eachother.

Aligning all of these elements as shown in FIG. 8 may be accomplished ina process that involves aligning the cameras 510, 520 to the slide 525and aligning the slide 525 to the X-Y stage. An example of such aprocess is:

-   -   A. Take an image of an array of biochemical experiments on the        slide using one of the cameras.    -   B. Calculate the angle, θ_(CAMERA-SLIDE), between the camera and        the slide using image alignment procedures. Store this angle for        later use.    -   C. Find the angle between the slide and the X-Y stage,        θ_(SLIDE-STAGE), using slide alignment procedures described        below.    -   D. Rotate the slide by the angle found in step (C) to align it        with the X-Y stage axes.    -   E. Rotate the camera by the sum of the angles found in steps (B)        and (C) to align it with the stage.    -   F. Repeat the slide alignment procedure of step (C) to obtain a        new slide mapping.    -   G. Repeat steps (B) and (C) to confirm all angles equal to zero.        If not, repeat entire process.    -   H. Repeat the entire process for the other cameras.

Alignment of the slide with the X-Y stage proceeds as described inconnection with FIG. 9, which illustrates reference frames used in slidealignment. In FIG. 9, reference frame 605 is aligned with an X-Y stagesuch as X-Y stage 137 in FIG. 1. Reference frame 610 is aligned with aslide such as slide 145 in FIG. 1. The two reference frames may berotated with respect to one another by a rotation stage such as rotationstage 135 in FIG. 1. In order to determine the required rotation angle(and offset and scale relationships), several points on the slide, suchas points “a” and “b” in FIG. 9, are measured in each reference frame.The location (x, y) of a point in the stage reference frame is knownfrom digital positioning commands issued to the stage.

The location (x′, y′) of a point in the slide reference frame isdetermined during image alignment procedures. If N points, indexed byi=1 to N are measured in both reference frames, then for point i one maywrite:x′ _(i) =a ₀₀ +a ₁₀ x _(i) +a ₀₁ y _(i) +a ₂₀ x _(i) ² +a ₁₁ x _(i) y_(i) +a ₀₂ y ² _(i)+Λy′ _(i) =b ₀ +b ₁₀ x _(i) +b ₀₁ y _(i) +b ₂₀ x _(i) ² +b ₁₁ x _(i) y_(i) +b ₀₂ y ² _(i)+Λ

The expansion above has been carried out up to second order. Expansionsto higher order, or in other coordinate systems, etc., may be usedwithout loss of generality. Next, an error term may be constructed:

$\chi^{2} = {\left( \frac{1}{N} \right){\sum\limits_{i = 1}^{N}\left\{ {\left( {x_{i}^{\prime} - x_{i}} \right)^{2} + \left( {y_{i}^{\prime} - y_{i}} \right)^{2}} \right\}}}$

Then, χ² is minimized to find coefficients a₀₀, a₁₀, a₀₁, . . . , b₀₀,b₁₀, b₀₁, . . . , etc. Finally the angle between reference frames may becalculated from the coefficients.

Once cameras, slide and stage are aligned, data acquisition may begin.The imaging systems of FIG. 1 have both static and dynamic imageadjustment capability. Static adjustments include magnification via zoomlens systems, rotation via a set of mechanical rotation stages, andwavelength selection via beam splitter and filter choices. Staticadjustments are made before slide scanning operations begin, whiledynamic adjustments can be made during a scanning operation.

Dynamic adjustments include small X and Y offset changes made via TDIpulse timing and galvo-driven rotation of a transparent flat plate,e.g., 110, rotation of a mirror, e.g., 111, or translation of a stage,e.g., 103. The dynamic adjustments may form part of an image-basedcontrol loop that corrects positioning error during scanning operations.The control loop involves acquiring images in cameras, clocking outimage data from the cameras, analyzing the data, calculating errorcorrections and adjusting X and Y offsets via TDI pulse timing and galvoplate angle.

As one skilled in the art will readily appreciate from the disclosure ofthe embodiments herein, processes, machines, manufacture, means,methods, or steps, presently existing or later to be developed thatperform substantially the same function or achieve substantially thesame result as the corresponding embodiments described herein may beutilized according to the present invention. Accordingly, the appendedclaims are intended to include within their scope such processes,machines, manufacture, means, methods, or steps.

The above description of illustrated embodiments of the systems andmethods is not intended to be exhaustive or to limit the systems andmethods to the precise form disclosed. While specific embodiments of,and examples for, the systems and methods are described herein forillustrative purposes, various equivalent modifications are possiblewithin the scope of the systems and methods, as those skilled in therelevant art will recognize. The teachings of the systems and methodsprovided herein can be applied to other systems and methods, not onlyfor the systems and methods described above.

In the following claims, the terms used should not be construed to limitthe systems and methods to the specific embodiments disclosed in thespecification and the claims, but should be construed to include allsystems that operate under the claims. Accordingly, the invention is notlimited by the disclosure, except as indicated by the claims.

What is claimed is:
 1. An imaging system comprising: a microscopeobjective; a camera configured to produce an image from scanning of asample along a scanning axis normal to an axis of rotational symmetry ofthe microscope objective; a lateral offset system along the axis ofrotational symmetry, the lateral offset system configured toindependently shift position of the image in the camera in a planenormal to the scanning axis; a rotational stage supporting the camera,the rotational stage being rotatable around the axis of rotationalsymmetry; and a zoom lens system configured to change a scale of theimage directed to the camera along the axis of rotational symmetry. 2.The imaging system of claim 1 wherein the zoom lens system is configuredto make static adjustments in magnification.
 3. The imaging system ofclaim 1 wherein the rotational stage is configured to make staticadjustments in rotation of the rotational stage around the axis ofrotational symmetry.
 4. The imaging system of claim 1 configured to makedynamic X and Y offset adjustments.
 5. The imaging system of claim 1wherein the camera is configured to operate in either TDI mode orfull-frame mode.
 6. The imaging system of claim 1 wherein the camera isconfigured to operate in full-frame mode.
 7. The imaging system of claim1 further comprising a positioning stage, the positioning stage beingtranslatable in an X direction and a Y direction in a plane normal tothe axis of rotational symmetry, wherein the sample is mounted on thepositioning stage.
 8. The imaging system of claim 7 wherein the samplecomprises a biochemical array mounted on the positioning stage.